WO2013087888A1

WO2013087888A1 - 3d microfluidic system having nested areas and a built-in reservoir, method for the preparing same, and uses thereof

Info

Publication number: WO2013087888A1
Application number: PCT/EP2012/075644
Authority: WO
Inventors: Thomas Berthelot; Hervé VOLLAND
Original assignee: Commissariat à l'énergie atomique et aux énergies alternatives
Priority date: 2011-12-15
Filing date: 2012-12-14
Publication date: 2013-06-20
Also published as: EP2790834A1; US20140349279A1

Abstract

The present invention relates to a three-dimensional (or 3D) microfluidic system including a plurality of layers (1, 3, 5, 7, 9) stacked on top of one another, characterized in that at least one of said layers consists of a first (3) and at least a second (10) portion which are separate from each other, wherein the second portion, which is porous and wettable by a solution of interest, is nested in a recess of the first portion, which is nonporous and/or non-wettable by said solution of interest, said system optionally comprising a built-in tank. The invention further relates to a method for manufacturing said system, and to the various uses thereof.

Description

3D MICROFLUIDIC SYSTEM WITH EMBEDDED ZONES

AND INTEGRATED TANK, ITS PREPARATION METHOD

AND ITS USES

DESCRIPTION

TECHNICAL AREA

The present invention relates to the field of diagnostic devices and in particular the field of such devices based on microfluidic systems.

Indeed, the present invention relates to a three-dimensional microfluidic system (or 3D) comprising hydrophilic zones, hydrophobic zones nested in each other and optionally an integrated reservoir and a method for preparing such a system.

STATE OF THE PRIOR ART

Immunological sensors in the form of dipsticks (or "dipsticks" in English) are commonly used for the detection of many biological parameters but also to enable the detection of pathogens ("1D lateral-flow Systems"). They are based on the lateral movement of fluids to be analyzed through strips or strips of paper. These systems, although widely used, have limitations in their bioanalytical and fluidic capabilities. In this format where the sample migrates in one dimension, it is difficult to multiply the number of biological parameters detected. Indeed, the multiplication of lines or zones of detection decreases the spatial resolution and can lead to a bad analysis of the result. From 2008, new detection devices have appeared and have been designated "3D microfluidic devices" (for "3D microfluidic devices"). The general concept of these new devices is to create hydrophilic microfluidic channels in a hydrophobic matrix and to stack successive layers using double-sided Scotch ^® . Indeed, in 2008, the group of Professor G. Withesides (Harvard University) developed, on the principle of immunological sensors in the form of strips, 3D microfluidic systems based on paper (and in a more general way based on a porous fibrous matrix) and SU-8 (photolithographic resin) [1-2]. Subsequently, similar systems but with a different embodiment were developed by the group of W. Shen [3] or the group of B. Lin [4].

These systems can especially be used for colorimetric, quantitative analyzes with integrated calibration. Thus, the international application WO 2011/000047 [5] envisages the use of a paper-based 3D microfluidic system comprising a plurality of hydrophilic test zones, in which the sample to be tested and standard samples containing a known quantity of the analyte to be assayed are deposited on different test areas and then the colorimetric intensity of the test sample is compared to the standard colorimetric range obtained from the standard samples. Note that in microfluidic systems as described in

[1] and [5], all the test areas are uniformly constituted by the same material.

So far, these 3D microfluidic systems all rely on a sandwich structure made by stacking distinct layers, at least one of which is a hydrophilic porous fibrous material made locally hydrophobic after a given treatment. Thus, each layer consists of the same base material locally having areas with physicochemical properties distinct from the (or) physicochemical treatment (s) undergone (s). More particularly, the technologies developed for producing these layers in the form of hydrophobic / hydrophilic matrices are derived from conventional lithographic techniques [1-2], "ink jet" techniques by printing hydrophobic molecules [3] or techniques wax printing by a laser printer [4]. Their concepts are therefore based on the development in a porous layer of a hydrophilic network surrounded by a matrix become hydrophobic, in all its thickness, following the chemical modification by impregnation of SU-8, wax or hydrophobic molecules. In particular, the international application WO 2010/003188 proposes a process for preparing a microfluidic system from a hydrophilic substrate [6]. The method comprises the steps of (i) hydrophobing the surface of the substrate, (ii) depositing on the hydrophobed surface a mask having aperture areas for defining the periphery of the microfluidic channels, and (iii) applying, at the level of exposed surface via the opening areas, a treatment with irradiations so as to make the surface hydrophilic. In step (i), the surface is rendered hydrophobic by absorption or adsorption of a solution of a hydrophobic substance as defined on page 4, lines 29 to 33 in a volatile solvent. The treatment with irradiations envisaged in particular is a plasma treatment or a corona treatment.

The techniques used to obtain these structures are therefore dependent on the thickness of the porous material used. These embodiments are in particular not applicable to thick porous fibrous matrices. Indeed, if the material to be treated to make it hydrophobic is too thick, it will be impossible to create channels in the matrix impregnated with photolithographic resin such as SU-8 by photolithography.

In the same way, it is impossible for the wax to permeate the entire thickness of the thick support. Even if the wax is sprayed at high temperature, its cooling will be faster than its diffusion. Thus, even if the matrix is heated to allow the wax to diffuse, it will result in a strong alteration of the design or even the capping of the hydrophilic channel in its thickness thus preventing the formation of a 3D microfluidic system.

Therefore, for thick matrices, it is impossible to use the photolithography / photolithographic resin approach, ink-jet printing of wax or hydrophobic molecules to obtain a completely hydrophobic matrix in contact with the hydrophilic zones, without causing a leak of the system in lateral hydrophilic tracks and / or without losing in resolution.

In addition, the use of photolithographic resin such as SU-8 and photolithographic processes are extremely expensive in terms of time and money as they are multi-step processes involving masking, revealing, rinsing steps, etc. .. For example, 500 ml of SU-8 are worth, in 2011, 5000 €.

It should be further noted that the hydrophilic matrix tracks as prepared by the aforementioned methods are brought into contact with other chemicals such as resin, solvents, wax vapor, rinse solvent, and this, before their use in the microfluidic system. This contacting can lead to possible contamination of the fluidic channel by by-products and cause artifacts when using the microfluidic system.

Finally, with the aforementioned methods, it is impossible to integrate in one and the same stage (between two double-sided Scotches ^® ) two or more porous hydrophilic materials of a different nature and / or thickness, such as, for example, introducing into the same stage a fluidic paper (cellulose) and fluidic fiberglass.

The inventors have therefore set themselves the goal of developing a method of manufacturing a microfluidic device 3D (i) independent of the thickness of the porous matrix, (ii) reducing the number of manufacturing steps compared to the processes of the prior art thus ensuring a saving of time, (iii) at low cost, (iv) having a good lateral resolution and (v) being able to integrate into the minus two fluidic systems based on porous materials of different chemical nature.

Finally, in the microfluidic devices 1D and 3D known until now, the deposit of samples is either by immersion of a part of the device in the sample, or by depositing the sample on the device. In the latter case, the device is inserted into a cassette which allows the deposition of a larger or smaller sample volume.

Also, in order to simultaneously detect different biological parameters using strips, plastic cassettes incorporating several have been developed. These cassettes use the distribution of the sample on the different strips and require a fractionation of the sample in as many aliquots as there are strips. Indeed, the previously described systems allow a capillary fractionation of the sample which can thus interact with different detection zones. This results in the analysis of small volumes for each parameter and therefore a decrease in the detection threshold or the need to use large sample volumes to overcome this fractionation.

The inventors have therefore set themselves the goal of developing a method of manufacturing a 3D microfluidic device having the advantages as previously listed and some embodiments of which also have a configuration allowing the analysis of several parameters without splitting. the sample in particular beyond a factor 2.

STATEMENT OF THE INVENTION

The present invention makes it possible to achieve the goal that the inventors have set themselves and to solve all or part of the technical problems of the prior art 3D microfluidic systems. More particularly, the system of the present invention and its method of preparation are based on the principle of marquetry. Indeed, the sandwich structure results from the stacking of layers in which the hydrophilic porous fibrous layer made locally hydrophobic is replaced by a mixed layer consisting of a hydrophobic support previously cut in the desired pattern, particularly by hand or by cutting with C0 ₂ laser printer, in which the porous hydrophilic material of interest is interlocked, the latter serving as a fluidic channel for the transfer of aqueous solutions and samples by capillary effect in the plane and / or in the thickness of the system final. It should be noted that the combination of a hydrophilic support with a porous hydrophobic material serving as a fluid channel is also conceivable in the context of the present invention.

The advantages of a 3D microfluidic system by "marquetry" according to the invention compared to the systems developed in the state of the art are numerous:

- cost reduction;

- Reduction of system development time, due to the absence of contacting steps, diffusion, annealing, exposure, revelation, washing, printing and / or heating;

- No limitation in the thickness of the material serving as a fluid channel unlike previous systems that can not use too thick matrices as previously explained;

- no obligation as to any treatment of the tracks of the matrix serving as fluidic channels to render them wettable by a solution of interest, these can be used in their "virgin" state ie without ever being put in contact with other products such as resin, solvents, wax vapor or rinse solvent;

great freedom as to the materials that can be used for the support part and for the part (s) of the fluidic channel type. Indeed, the fact that the support part and the (or) part (s) of the fluidic channel type are nested and therefore dissociable from each other makes it possible to envisage many forms of implementation impossible with the microfluidic systems of the state of the art. First, the support portion and the fluidic channel type portion (s) may be prepared from different materials. There is no limitation as to the materials that can be used for these different parts, unlike the systems of the state of the art in which the initial layer must be in a single hydrophilic (or hydrophobic) material that can be rendered hydrophobic (or hydrophilic). following a given treatment.

Similarly, when the microfluidic system according to the invention comprises several parts of fluidic channel type on the same layer, all or only some of these parts may be of a material, identical or different. Also, unlike systems of the prior art, the present invention allows to introduce, in the same stage, 2 to several types of porous matrix of different chemical nature, such as, for example, paper and glass fiber.

This latitude makes it possible not only to adapt the physicochemistry of the fluidic channels to the molecules to be analyzed, but also to envisage the preparation of 3D microfluidic systems according to the invention "à la carte". For example, it is possible to fit in a previously prepared support, one (or more) part (s) of fluidic channel type adapted (s) to one (or more) molecule (s) of interest, according to the wish of the user and at the moment when the user expresses it.

Thus, the present invention relates to a three-dimensional (or 3D) microfluidic system comprising a plurality of layers stacked one on top of the other, wherein at least one of said layers consists of a ^1st and at least a 2 ^nd parts, distinct from each other, with the 2 ^nd part porous and wettable with a solution of interest fitting into a recess of the ^era nonporous portion and / or non-wettable by said solution of interest, said 2 ^nd portion serving as a fluid channel for transferring said solution of interest by capillarity effect in the plane and / or the thickness of said system.

In the microfluidic system according to the invention, the l ^st part and

(or) 2 ^{nd (s)} part (s) are chemically different due in particular to their difference in wettability and / or porosity.

In the microfluidic system according to the invention, the porous and wettable part 2 ^nd by a solution of interest is previously cut and is in a form adapted to its nesting in the recess of the ^1st part.

In addition, the ^era part constitutes the support of the layer such as described above and (or) 2 ^{nd (s)} part (s) form (s) the fluid channel via the stack of layers that comprises the microfluidic system according to the invention. Thus, the terms ^"ere partie" and "support portion" are equivalent and used interchangeably. Similarly, the terms "2 ^nd part" and "fluidic channel-like portion" are equivalent and used interchangeably.

As previously explained, at least one layer of the 3D microfluidic system according to the invention may comprise a single 2 ^nd part porous and wettable with a solution of interest or at least two 2 ^ndes parts, at least three ^ndes two parts, at least four 2 ^nportions , porous and wettable by a solution of interest, of the same or different nature and of identical or different form, all of these 2 ^nthe parts nested in as many recesses as 2 ^nd parts, present in level of the support part of the layer.

In a particular embodiment, it is possible to maintain at least one of the recesses of the first ^uncompleted portion. Thus, the microfluidic system of this particular form of implementation includes a plurality of layers stacked on each other, of which at least one layer consists of a l ^st part and at least one 2 ^nd part, distinct from each other, with the 2 ^nd porous portion and wettable with a solution of interest fitting into a recess of the ^era nonporous portion and / or non-wettable by said solution of interest, the said ^ere partie further having at least one recess unmet and said 2 ^nd part serving as a fluidic channel ensuring the transfer of said solution of interest by capillarity effect in the plane and / or the thickness of said system.

Advantageously, in this particular implementation, the microfluidic system may comprise at least one layer of the constitutée ^ere partie having at least one non-filled recess and at least one 2 ^nd part as defined above. In particular, the microfluidic system may comprise (i) at least one layer of the constitutée ^ere partie having an unmet recess and a 2 ^nd part as defined above, (ii) at least one layer of a constitutée ^ere the portion having an unmet recess and two 2 ^ndes parts as defined above and / or (iii) at least one layer of the constitutée ^ere partie having an unmet recess and three parts ^ndes 2 as defined above . Thus, the 3D microfluidic system according to the invention may comprise at least one layer (i) and at least one layer (ii), at least one layer (i) and at least one layer (iii) or at least one layer (ii). ) and at least one layer (iii). Similarly, the 3D microfluidic system according to the invention may comprise at least one layer (i), at least one layer (ii) and at least one layer (iii).

When the microfluidic system according to the invention has several layers each comprising at least one uncompleted recess and in particular a single unfilled recess, the stack of these layers constitutes a cannula and a reservoir integrated in the system. To do this, the recesses of these different layers are in fluidic continuity with each other. vis-à-vis others. Advantageously, all or a portion of the recesses are centered on a common axis. Advantageously, all the recesses or some of the latter are centered on a common axis and have a cross section with respect to the plane of the same shape layer, the thickness being dependent on the thickness of the ^ere partie wherein they been prepared.

In the context of the present invention means, for each tank, a cavity defined by the recess of the portion of the ^st ^st layer and delimited by at least the next layer. The cavity can be delimited by the following single layer: in this case, the reservoir is located at the layer of the upper end of the microfluidic system. Alternatively, the cavity is delimited by both the next layer and the previous layer: in this case, the tank is said to be integrated in the system. The next layer and / or the preceding layer has at least one recess possibly filled at the recess forming the reservoir, the two recesses being in fluid contact with each other. In the case of a non-integrated reservoir, only the next layer comprises such a recess in which is nested a 2 ^nde part as previously defined. In the case of an integrated reservoir, the preceding layer comprises at least one recess that can be filled, partially filled or not filled by a 2 ^nde portion as defined above. In this embodiment, the next layer may also comprise at least one recess may be filled, partially filled or not filled by a 2 ^nd part as defined above, the recesses of the next and previous layers or portions thereof that can be centered on a common axis or on the contrary, not centered on a common axis. As a variant of this embodiment, the following layer may not comprise a recess in fluidic contact with the reservoir and, in this case, the preceding layer comprises at least two distinct recesses which may be, independently of one of the other, filled, partially filled or not filled by a 2 ^nd part as previously defined. In this particular embodiment, the solution of interest "descends" to the reservoir via a cannula or fluidic channel system and "rises" into the upper layers via a separate cannula or fluid channel system.

Advantageously, the tank used in a microfluidic system according to the invention is in fluid contact with at least one fluidic channel as defined above and, to this end, in the recess of the ^era portion corresponding to said reservoir, may be an element made of a hydrophilic and porous material (case of a solution of hydrophilic interest) or a hydrophobic and porous material (case of a solution of hydrophobic interest). This element does not constitute a 2 ^nd part as defined above as it does not completely fill the recess in the l ^st part. The l ^st and 2 ^{nd (s)} parts of at least one layer of the microfluidic system according to the invention are in particular defined in relation to their wettability or non-wettability vis-à-vis of a solution of interest.

The wettability is defined by the contact angle (or connection angle) that forms a drop of the solution of interest with the portion at the deposition site of this drop.

Thus, when it is specified that the 2 ^nd part (s) is (are) wettable with respect to the solution of interest, this generally means that drop of this solution deposited will form with respect to the deposition site generally a contact angle having a value less than 70 ° and in particular less than 60 ° while, for the ^era optionally part nonwettable, this means that the angle of contact formed between a drop of the solution and this part generally has a value greater than 90 ° and in particular greater than 95 °. From a practical point of view, this means that, when the solution of interest is deposited on the 2 ^nd part (s), it remains at this level without going into the ^1st non-wettable part .

From a chemical point of view, a liquid will wet the substrate that represents the part of the microfluidic system if it has a chemical affinity vis-à-vis it. Thus a hydrophilic (or hydrophobic) substrate will be wettable vis-à-vis the hydrophilic (or hydrophobic) liquids.

It should be noted that the definition of wettability as given above relates to the conventional definition of wettability with respect to a liquid on a flat surface. In the context of the present invention, the chemical nature and in particular the wettability of the surface of the (or 2 ⁾ part (s) is involved in the transfer of the solution of interest which is done by capillarity but not only because the solution also passes through the open pores of the material of this (or these) 2 ^{nde (s)} part (s). Thus, the 2 ^{nd (s)} part (s) can also be defined as permeable (s) to the solution of interest. In the end, the (or 2 ⁾ part (s) constitute (s) a porous system through which the solution of interest circulates, this circulation being faster than the hydrophilicity / hydrophobicity of the material. porous is compatible with the hydrophilicity / hydrophobicity of the solution of interest.

In the context of the present invention, "solution of interest" is understood to mean any natural or synthetic hydrophilic or hydrophobic solution that one wishes to analyze and / or purify on a 3D microfluidic system according to the present invention. It should be emphasized that the implementation solution in the interest of the present invention should be neither a solvent of the ^era party nor a solvent of any of the two parts of ^emes 3D microfluidic system.

Thus, this solution of interest can be a biological fluid; a vegetable fluid such as sap, nectar and root exudate; a sample in a culture medium or in a biological culture reactor as a culture cellular higher eukaryotes, yeasts, fungi or algae; a liquid obtained from one (or more) animal (s) cell (s) or plant (s); a liquid obtained from an animal or plant tissue; a sample taken from a food matrix; a sample taken from a chemical reactor; town water, river, pond, lake, sea, air-cooled towers; a sample from a liquid industrial effluent; waste water, particularly from intensive livestock farms or chemical, pharmaceutical, cosmetic or nuclear industries; a pharmaceutical product; a cosmetic product; a perfume ; a soil sample or a mixture thereof.

The biological fluid is preferably selected from the group consisting of blood such as whole blood or anti-coagulated whole blood, blood serum, blood plasma, lymph, saliva, sputum, tears, sweat, sperm, urine, stool, milk, cerebrospinal fluid, interstitial fluid, isolated bone marrow fluid, mucus or fluid from the respiratory tract, intestinal or genitourinary tract, cell extracts, extracts from tissues and organ extracts. Thus, the biological fluid may be any fluid naturally secreted or excreted from a human or animal body or any fluid recovered from a human or animal body by any technique known to those skilled in the art such as extraction. , a sample or a wash. The steps of recovery and isolation of these different fluids from the human body or animal are performed prior to the implementation of the method according to the invention.

Similarly, if one of the samples envisaged does not allow to implement it with a 3D microfluidic system according to the present invention, for example because of its particular solid nature, its concentration or the elements it contains such as solid residues interfering molecules, this implementation and in particular the detection method as defined below furthermore comprises a preliminary step of preparation of the solution of interest with possible dissolution of the sample by techniques known to those skilled in the art such as filtration, precipitation, dilution, distillation, mixing, concentration, lysis, etc.

Different forms of implementation on the part of the ^era at least one layer of 3D microfluidic system according to the invention are possible. Indeed, the latter can be:

- porous and non-wettable by the solution of interest;

- non-porous and wettable by the solution of interest; or

- non-porous and non-wettable by the solution of interest.

Thus, to a solution of hydrophilic interest, ^ere the part (ie the support portion) of at least one layer, in particular one (or more) layer (s) and advantageously all of the system layers microfluidic according to the invention can be:

- porous and hydrophobic;

- non-porous and hydrophilic; or

- non-porous and hydrophobic;

the (or) 2 ^{nd (s)} part (s) being, in the case of a solution of hydrophilic interest, absorbent (s) and porous (s).

Advantageously, for a solution of hydrophilic interest, the support portion of at least one layer, in particular one (or more) layer (s) and advantageously all the layers of the microfluidic system according to the invention is chosen in the group consisting of a porous and hydrophobic polymeric film; a hydrophobic porous membrane, gel or resin; a non-porous hydrophilic or hydrophobic polymeric film and a non-porous hydrophilic or hydrophobic membrane or resin.

In particular, by way of illustrative and nonlimiting examples, for a solution of hydrophilic interest, the support portion of at least one layer, in particular one (or more) layer (s) and preferably all layers of the microfluidic system according to the invention is selected from the group consisting of a porous or non-porous film of polyethylene terephthalate (PET); a porous or non-porous membrane of polyethylene (PE); a porous or non-porous polypropylene (PP) membrane; a porous or non-porous film, or a porous or non-porous, fluorine-containing membrane such as a porous or non-porous film, or a porous or non-porous membrane of hydrophobic polyvinylidene fluoride (PVDF); a porous or non-porous membrane of polytetrafluoroethylene (PTFE); a porous or non-porous copolymer film comprising vinylidene fluoride and tetrafluoroethylene; a porous or non-porous copolymer film comprising vinylidene fluoride and hexafluoropropylene; a porous or non-porous film of polymethyl methacrylate (PMMA); a porous or non-porous film of poly (n-butyl acetate); a porous or non-porous film of poly (benzyl methacrylate); a porous or non-porous film of poly (chlorotrifluoroethylene); a porous membrane and, in particular a porous ion exchange membrane, functionalized with hydrophobic groups; a styrenic polymer such as an advantageously porous polystyrene resin; a porous or non-porous film of polyacrylonitrile; a porous or non-porous film of polymethacrylonitrile; a film, porous or nonporous, polyimide such as Kapton ^® and mixtures thereof. By "hydrophobic group" is preferably meant, in the context of the present invention, a fluorinated group; an aryl group optionally substituted with one or more fluorine atoms and in particular a phenyl group optionally substituted with one or more fluorine atoms; and an alkyl group optionally substituted with one (or more) fluorine atom (s).

Similarly, to a solution of hydrophilic interest, (or) 2 ^{nd (s)} part (s) hydrophilic (s) and porous (s) is (are) advantageously selected (s) from the group consisting of a material Hydrophilic fibrous, tissue, hydrophilic film porous, a hydrophilic gel or a porous membrane bearing or functionalized with hydrophilic groups. By "hydrophilic group" is meant in the context of the present invention, a group selected from a hydroxyl group, a carbonyl group, a trialkoxysilane group, an anionic group, a cationic group, a tertiary amine group, an epoxy group, an ester group, an amide group, an anhydride acid group, a phosphonic acid group, a sulfonic acid group, an ammonium group and any conjugate bases thereof.

More particularly, by way of illustrative and non-limiting examples, for a hydrophilic interest solution, the (or) 2 ^{nd (s)} part (s) hydrophilic (s) and porous (s) is (are) advantageously selected (s) in the group consisting of paper including cellulosic nature; cotton paper; agarose; gelatin; cellulose; methylcellulose; carboxymethylcellulose; nitrocellulose; cellulose acetate ester; an alginate; a polyolefin; a porous membrane and, in particular a porous ion exchange membrane, functionalized, advantageously by radiografting, with hydrophilic groups such as a NAFION membrane; a membrane-type Sephadex resin or a PVDF membrane; a fiberglass fabric; a polyacrylamide gel; a sepharose gel or a mixture thereof.

As a variant, for a solution of hydrophobic interest, the support part of at least one layer, in particular of one (or more) layer (s) and advantageously of all the layers of the microfluidic system according to the invention can be :

- porous and hydrophilic;

- non-porous and hydrophobic; or

- non-porous and hydrophilic; the (or the) 2 ^{n els)} part (s) being, in the case of a hydrophobic solution, hydrophobic (s) and porous (s).

Advantageously, for a solution of hydrophobic interest, the support portion of one (or more) layer (s) and advantageously all the layers of the microfluidic system according to the invention is chosen from the group consisting of a polymer film porous and hydrophilic; a membrane, a gel or a porous hydrophilic resin; a non-porous hydrophilic or hydrophobic polymeric film and a non-porous hydrophilic or hydrophobic membrane or resin.

In particular, by way of illustrative and nonlimiting examples, for a solution of hydrophobic interest, the support portion of at least one layer, in particular one (or more) layer (s) and preferably all layers of the microfluidic system according to the invention are selected from the group consisting of paper; cotton paper; agarose; gelatin; cellulose; methylcellulose; carboxymethylcellulose; nitrocellulose; cellulose acetate ester; an alginate; a polyolefin; a membrane and, in particular an ion exchange membrane, functionalized, advantageously by radiografting, with hydrophilic groups such as a NAFION membrane; a membrane-type Sephadex resin or a PVDF membrane; a fiberglass fabric; a non-porous film of polyethylene terephthalate (PET); a non-porous polyethylene (PE) membrane; a non-porous polypropylene (PP) membrane; a non-porous film of polyimide such as Kapton ^®; a polyacrylamide gel; a sepharose gel or a mixture thereof.

In particular, by way of illustrative and non-limiting examples, for a hydrophobic solution of interest, (or) 2 ^{nd (s)} part (s) hydrophobic (s) and porous (s) is (are) chosen ( s) in the group consisting of a hydrophobic fibrous material, a hydrophobic fabric, a porous hydrophobic film, a gel or a porous membrane bearing or functionalized with hydrophobic groups.

More particularly, by way of illustrative and non-limiting examples, for a hydrophobic solution of interest, (or) 2 ^{nd (s)} part (s) hydrophobic (s) and porous (s) is (are) advantageously selected (s) in the group consisting of paper made hydrophobic by treatment, a porous film of polyethylene terephthalate (PET); a porous polyethylene (PE) membrane; a porous polypropylene (PP) membrane; a porous film or a porous membrane containing fluorine such as a porous film or a porous membrane of hydrophobic polyvinylidene fluoride (PVDF); a porous polytetrafluoroethylene (PTFE) membrane; a porous copolymer film comprising vinylidene fluoride and tetrafluoroethylene; a porous copolymer film comprising vinylidene fluoride and hexafluoropropylene; a polyacrylamide gel; a porous polymethyl methacrylate film (PMMA); a porous film of poly (n-butyl acetate); a porous film of poly (benzyl methacrylate); a porous film of poly (chlorotrifluoroethylene); a porous membrane and, in particular a porous ion exchange membrane, functionalized with hydrophobic groups; a styrenic porous polymer such as a porous polystyrene resin; a porous polyacrylonitrile film; a porous film of polymethacrylonitrile and a mixture thereof.

That ^ere the part of a layer 3D of the microfluidic system according to the invention is hydrophobic or hydrophilic, it may present on at least one adhesive face in direct contact with another layer of the system. Such an adhesive face may consist of a PET transparency adhesive on both sides, glued on a ^1st part of a layer. This variant makes it possible to join two layers of the system together and / or to guarantee the seal between these two layers. The (or) 2 ^"e (s) part (s) of a layer 3D microfluidic system according to the invention is (are) t hydrophobic (s) or hydrophilic (s), it (s) bit (Fri. at least one reagent or compound capable of detecting and / or trapping a constituent or an analyte present in a solution of interest, since, as explained hereinafter, the 3D microfluidic system according to the invention may be used in methods for detecting and / or purification. in other words, at least one reactant or compound is incorporated in the porous material of at least one 2 ^nd part implemented in the context of the present invention.

When the material of this 2 ^nd portion comprises at least one reagent or compound, these can be either distributed throughout the volume of the material or located in a specific area of the material. Advantageously, the reagents or compounds may be on the surface of the pores or channels of the material. The reagents or compounds can be adsorbed on the surface of the pores or channels of this material and / or bound to this surface by non-covalent bonds (hydrogen bonds or ionic bonds) and / or by covalent bonds.

When the bond between the reactant or compound and the material of the 2 ^nd portion is low, the reagent or the compound can be led to lower layers by the flow of the solution of interest, once the latter is placed on the system microfluidics.

On the contrary, in the case of a strong bond, the reagent or the compound remains in this material even in the presence of the solution of interest and the detection and / or trapping take place in this same layer. It is the same in the case of a reagent or a compound not covalently bound but too voluminous with respect to the pores and channels of the fluidic channel portion of the subsequent lower layer. For fastening, by means of covalent bonds, the reactive compound or the material of the ^2nd part, various methods known to those skilled in the art whether or not using spacer arms. are usable. In addition, the experimental part hereinafter describes a process using cleavable aryl diazonium salts and based on the process described in International Application WO 2008/078052 [7] and a method using aryl azide salts for fixing reagents or compounds on a material of the 2 ^nd part.

In addition, the 3D microfluidic system according to the invention may comprise at least 2 layers, at least 3 layers, at least 4 layers, at least 5 layers, at least 10 layers, or even at least 50 layers, all or part of these layers. may have a ^lre and at least a 2 ^nde parts as previously defined.

In the present invention, the nomenclature "upper layers" and "lower layers" is assessed relative to the support on which the microfluidic system according to the invention is optionally placed. Thus, the layer of the lower end corresponds to the layer in direct contact with this support, the layer of the upper end corresponding to the furthest layer vis-à-vis the support.

These layers may have an identical thickness or different from each other. Advantageously, the thickness of each layer is between 0.1 μιη and 20 mm, in particular between 0.5 μηι and 5 mm, typically between 1 μιη and 1 mm, in particular between 2 μιτι and 400 μηι and, more particularly, between 5 μιη and 200 μιη thus making it possible to obtain a 3D microfluidic system having a thickness of between 2 μιη and 100 mm, typically between 5 μιη and 50 mm, in particular between 10 μιη and 25 mm and, in particular, between 40 μιη and 10 μιη. mm. It should be noted that, for the same layer of the 3D microfluidic system according to the invention, the thickness of the (or) 2 ^{nd (s)} part (s) should be adjusted according to the thickness of the l ^st part. Advantageously, for the same layer, the l ^st part and (or) 2 ^{nd (s)} part (s) have substantially identical thicknesses. Advantageously, all the layers included in the 3D microfluidic system according to the invention exhibit the ^st part and at least one 2 ^nd part as defined above. In some form of implementation, among the layers included in the 3D microfluidic system according to the invention having the ^ere partie and at least one 2 ^nd part as defined above, one or more of these layers may comprise at least one recess in the ^ere partie, unmet.

It is also possible that some of the layers included in the 3D microfluidic system according to the invention have a support part with one (or more) recess (s) without, in the latter, a 2 ^nd portion of fluidic channel type such as previously defined is nested. In other words, at least one layer of the stack of layers constituting the microfluidic system according to the invention consists solely of a non-porous support and / or non-wettable by a solution of interest and hollowed out at the level of one (or more) defined area (s). It is clear that this (or these) recess (s) are part of the (or) channel (channels) fluidic (s) that includes the microfluidic system.

When such a layer is between two layers comprising a ^1st part and at least a 2 ^nd part as previously defined, this layer could then be considered as a switch or a fluidic selection or distribution system. Indeed, as long as the 2 ^ndivided parts of the layers, separated by the layer without 2 ^nde part, are not in contact, there is discontinuity in the fluidic system and the liquid can not pass, as explained in paragraph 84 of [1]. On the other hand, if a mechanical action of pressure or crushing on this zone is carried out as described in paragraph 83 of [1], the two stages are then brought into contact and the fluidics system is then connected and the solution of interest can pass.

A layer having a support portion with one (or more) recess (s) without, in the latter, a 2 ^nd portion of fluidic channel type as defined above is nested is advantageously present in the stack of layers that constitutes the 3D microfluidic system according to the present invention, among the upper and lower layers of this stack. More particularly, such a layer is the l ^st layer of this stack (ie the layer on which the solution of interest is deposited) and / or the final layer of said stack. In this form of implementation, the (or) recess (s) which the l ^st layer (or layer of the upper end) serve (s) solution of interest tank and the last layer (or layer the lower end) can be used to maintain the layer just above it and in particular the (or) 2 ^{nde <s)} part (s) that includes it. In this form of implementation, the reservoir is located at the top of the device (ie at the l ^st layer), whereas in the previously proposed variant implementing one or more layers having the ^era part, at least a 2 ^nde part as defined above and at least one unfinished recess at said ^1st part, the reservoir is preferably at lower layers ie is integrated in the structure of the system.

The use of layers with particular properties in terms of the number of 2 ⁿ parts or in terms of thickness, porosity and / or functionalization of the material constituting these 2 ^nd parts makes it possible to give the layers a particular function. Some of these layers may have a function in the distribution and division of the solution of interest, in the analysis, in the detection and / or in the purification.

Thus, the 3D microfluidic system according to the invention may in particular have at least one layer of which at least a 2 ^nde part as defined above and in particular whose 2 ^nde part as previously defined plays the role of biological bin. The latter makes it possible to recover all or part of the solution of interest once the detection, quantification and / or purification of a given analyte has been carried out. Also general and as described below, the shape of the recesses made in the ^era of part of the inventive system layers and hence that of the (or) 2 ^{nd <s)} part (s) optionally filling are very varied. In a particular implementation, the 3D microfluidic system according to the invention can in particular have at least one layer which at least one 2 ^nd part as defined above and in particular with the 2 ^nd part as defined above has the shape spiral or double spiral.

Similarly, it may be advantageous to control the thickness of at least one layer and / or the porosity of at least a 2 ^nd portion of at least one layer of the 3D microfluidic system according to the present invention. Those skilled in the art know various techniques for controlling the porosity of a hydrophilic or hydrophobic porous material as previously described. This control is notably implemented at the time of preparation of the hydrophilic and porous material (or of the hydrophobic and porous material). It may depend on the conditions in particular weaving conditions in the context of a woven material and / or reagents used during this preparation and in particular the addition of blowing agents in the reaction medium.

As regards the interest of such a control, mention may be made of the example of the filtration of a solution of interest in the form of a complex medium comprising molecules, such as proteins, peptides and modified sugars. or not, organic molecules entering the different signaling or metabolic pathways etc., in solution, cells but also cell debris membrane debris type. The 3D microfluidic system according to the invention can, for example, be used to detect the presence of a particular protein in this complex mixture. Also, in order to avoid measuring artifacts, the complex can be deposited on the medium 2 ^nd portion of the first layer which has a thickness and a controlled porosity (less than the size of the cells and cellular debris) to purify the sample and get the output of this part 2 ^does, only the medium containing molecules, such as proteins, peptides, sugars, modified or unmodified, organic molecules falling within the various signaling pathways or metabolic etc., cells and cellular debris being trapped in the material constituting this part.

Another example is the deposition of a solution of interest containing previously lysed cells. Depositing the solution on the 2 ^nd portion of the first layer which has a thickness and a controlled porosity to purify the sample bearing trapped in the paper membrane debris and organelles and will leave just pass the internal contents to cells such as proteins, DNAs or different metabolites.

Likewise, it is possible for the same 3D microfluidic system to ^have parts made of porous materials that can be ^wetted by a different solution of interest, which make it possible to fix molecules or organic materials of different types on the same system. As examples, there may be areas or 2 ^ndes parts containing porous wettable materials by a particular solution of interest, different because of their chemical nature, due to their thickness or due to their porosity. Indeed in the same 3D microfluidic system one may have to analyze at the same time different types of biological molecules such as proteins and cellular elements. However, these different elements to be analyzed will not have the same physico-chemical properties vis-à-vis the porous matrix. Indeed, some may be adsorbed by electrostatic effect preventing any migration into the system and making analysis impossible if it is this element that we want to analyze or quantify. This is particularly the case of certain bacteria that are adsorbed very strongly in nitrocellulose membranes thus preventing their migration within the system and rendering it ineffective for detection. On the contrary, if we want to get rid of this element for analysis we can then use this phenomenon. Therefore, if it is desired to analyze a protein and a bacterium with the same 3D microfluidic system according to the invention and if the porous matrix used for the protein can not be used for the bacterium (for the reasons explained above), it is necessary to use conduction channels of different natures within the same floor ie of the same layer.

In a particular embodiment of the microfluidic system according to the present invention, the latter has, between at least two consecutive layers, an element capable of joining these two layers together and / or to guarantee the seal between these two layers. Advantageously, such an element, identical or different, is present between all the layers that presents the microfluidic system according to the present invention.

This element may be in the form of a double-sided Scotch ^® , stretch film of the Saran microwave stretch film type or a derivative of a supported adhesion primer.

When double-sided Scotch ^® or stretch film is implemented, the latter has at least one recess and in particular one (or more) recess (s) to ensure the continuity of the fluid channel between the two layers it is secured. Advantageously, one (or more) of these recesses is (are) filled (s) by a porous and wettable material with a solution of interest, identical to or different from the material of one of the 2 ^{nd (s)} part (s) layers that it solidarises together. Alternatively, this (or these) recess (s) is (are) left (s) in the state. However, as previously explained for the layers of the system that may have a support portion with at least one recess without any 2 ^nde part of fluidic channel type as defined above is nested, it may be necessary to act mechanically by pressure or by crushing on unfilled hollow areas, to ensure the fluidic continuity of the layers separated by such scotch ^® or stretch film. The concept of "derivative of a supported adhesion primer" used in the context of the present invention is directly related to the invention described in the international application WO 2009/121944 [8]. Indeed, this application relates to a method for assembling two surfaces together. In the context of the present invention, these two surfaces are the lower surface of a layer and the upper surface of the consecutive layer. The derivative of a supported adhesion primer bridges these two surfaces by means of covalent bonds. Typically, such adhesion primer supported is present on all or part of the ^era portion of at least one of the two layers. It is advantageously a supported cleavable aryl salt and, in particular, a supported cleavable aryl diazonium salt of formula (I) below:

(surface) - (B) _n -RN ₂ ⁺ , A ^" (I)

in which :

surface representing the surface of a ^1st part of a layer of a 3D microfluidic system according to the invention,

- (B) _n represents a binding agent,

n is 0 or 1,

- A represents a monovalent anion and

- R represents an aryl group.

B may represent a single entity, at least two identical or different entities or even a primer as described in [8]. Similarly, all the variants and embodiments envisaged in [8] for A and R can be used in the context of the present invention. The present invention also relates to a device comprising a microfluidic system as defined above. Such a device may be in the form of a plate, a box or a plastic or ceramic housing in which () the 3D microfluidic system according to the invention is placed. The 3D microfluidic system or the device according to the invention can be embedded in a suitable packaging including instructions for use but also in a more complicated system where the consumable can be, for example, the 3D microfluidic system or the device according to the invention. The present invention also relates to a method of manufacturing a 3D microfluidic system as defined above, said method comprising the steps of:

ai) preparing, in a layer of a non-porous material and / or non-wettable by a solution of interest in particular as defined above, a recess;

bi) cutting, in a layer of a porous material and wettable by said solution of interest in particular as defined above, at least one form consistent with the recess prepared in step (ai);

Ci) nesting in the recess prepared in step (ai) the cut form in step (bi), whereby a layer of said microfluidic system 3D is obtained;

di) optionally repeat steps (ai), (bi) and (ci); and ei) assembling the different layers of the 3D microfluidic system. Step (I) of the process is to prepare the ^era part of a layer of 3D microfluidic system. This recess can be obtained by any cutting technique known to those skilled in the art. Advantageously, the latter is chosen from a selective chemical etching cut, a physical cut of the reactive ion etching (RIE) type, a manual cut such as embossing and a laser cut such as a pulsed laser including a YAG type source (acronym for "Yttrium Aluminum Garnet" or yttrium garnet aluminum) or a continuous laser including a source laser C0 ₂ . These lasers make it possible to obtain a resolution of the order of 5 to 10 μιτι. During step (a1), at least one other recess can be prepared in the layer of a non-porous and / or non-wettable material by a solution of interest. This recess can be subsequently filled during the implementation of steps (bi) and (ci) or, conversely, remained in the uncapped state.

During step (ai), the recess (or recesses) prepared may have various forms. As conceivable forms, there may be mentioned a recess whose section relative to the plane of the layer is circular, oval, square, rectangular, triangular, star-shaped, cross, rod, spiral or double spiral . On the same layer, two recesses may have the same or different shape. Similarly, two materials filling two recesses each belonging to two successive layers and fluid continuity of each other may have a section relative to the plane of the layer of identical or different shape.

The use of a recess whose cross-section relative to the plane of the layer is in a spiral or double spiral may have some advantage in terms of a layer which at least one 2 ^nd part is implementation in detection and / or purification methods. Indeed, such a configuration makes it possible to carry out several detection zones (ie zones comprising at least one reagent or compound, capable of detecting and / or trapping a constituent or an analyte present in a solution of interest). Thus, several constituents or analytes can be identified from the same sample of solution of interest without requiring fractionation of said sample.

Step (bi) of the method according to the present invention consists in preparing at least a 2 ^nd portion of a layer of the 3D microfluidic system according to the present invention. By "shape in conformity with the recess" is meant not only a shape fitting perfectly into the recess prepared in step (a1) but also a shape whose thickness is substantially equal to the thickness of the layer implemented in step (ai). Advantageously, the cutout implemented in step (b ₂ ) is also chosen from a cutout by selective chemical etching, a physical cut reactive ion etching type, manual cutting and laser cutting as previously defined.

The steps (ai), (bi) and (below) are repeated as many times as 3D microfluidic system includes layers with the ^era part and at least one 2 ^nd part as defined above.

The layer obtained following the nesting step (ci) can be defined as a 2D microfluidic system as integrated in the plane of the support system.

The nesting of step (ci) and the assembly of step (ei) can be carried out manually or in an automated manner.

During step (ei), the layers to be assembled may comprise not only one (or more) layer (s) as (s) prepared (s) by implementing steps (ai), (bi) and ( ci) but also one (or more) layer (s) prepared (s) by the sole implementation of step (ai) (ie one (or more) layer (s) constituted (s) of a material not porous and / or non-wettable for a liquid of interest and having one (or more) obviously left in the state) and / or one (or more) layer (s) of such adhesive ( s) than previously defined.

In the particular embodiment in which the 3D microfluidic system according to the present invention comprises a derivative of a supported adhesion primer, the method of preparation comprises the steps of:

ai ') preparing, in a layer of a non-porous material and / or non-wettable by a solution of interest especially as defined above and whose surface has a supported adhesion primer and in particular the cleavable aryl salt supported , a recess; bi) cutting, in a layer of a porous material and wettable by said solution of interest in particular as defined above, at least one form consistent with the recess prepared in step (a ^);

Ci) nesting in the recess prepared in step (ai ') the form cut in step (bi), whereby a layer of said microfluidic system 3D is obtained;

di) optionally repeat the steps (a ^), (bi) and (ci); and ei ') assembling the various layers of the 3D microfluidic system by subjecting the surface of the non-porous and / or non-wettable material with a solution of interest to non-electrochemical conditions to obtain, from the supported adhesion primer and in particular from the cleavable aryl salt supported on said surface, at least one radical and / or ionic entity and bringing into contact said surface having at least one radical and / or ionic entity thus obtained with the surface of the consecutive layer.

During step (ai '), the functionalization of the surface of the material by a supported adhesion primer can be done before or after the recess of the material.

Steps (bi), (ci) and (di) are as previously defined. Similarly, the implementation forms previously envisaged for step (ai) apply mutatis mutandis to step (ai ').

The non-electrochemical conditions that can be used during step (ei ') are as defined in [8]. Note however that it is desirable that these conditions do not implement solutions likely to act on the materials constituting the layers of the microfluidic system.

Thus, advantageously, the non-electrochemical conditions that can be used during step (ei ') are non-electrochemical photochemical conditions and consist in subjecting the supported adhesion primer and in particular the cleavable salt supported to irradiation (or insolation) in the UV or visible domain. The wavelength used in step (ei ') of the process will be chosen without any inventive effort, depending on the adhesion primer and in particular the cleavable aryl salt used. Any wavelength belonging to the UV domain or to the visible range can be used in the context of the present invention. Advantageously, the wavelength applied is between 300 and 700 nm, in particular between 350 and 600 nm and, in particular, between 380 and 550 nm.

The microfluidic system offers a great diversity in conceivable configurations. In the simplest configuration, the solution of interest is deposited on the layer of the ^era system and is recovered at a lower layer including at the last layer. When such a device is used in detection or purification, the detection can also be implemented at a lower layer and in particular at the level of the last layer.

Alternatively, and especially when the microfluidic system has an integrated reservoir therein, the solution of interest may be in contact or pass through several layers multiple times, the ^era times to go to the tank in an inner layer system (meaning down), a 2 ^nd time to travel from the reservoir to the analysis or detection layer, the latter advantageously being the l ^st layer system (upward direction), and a ^3rd and final time to move the layer analysis up to a lower layer and in particular the last layer which plays a role "trash" but which also has a role in the capillarity.

Whatever the configuration envisaged, it is important to note that the routing of the solution of interest in or through the layers of the system is only by gravity and / or capillarity, no particular pump type element mechanical n ' is necessary for this routing. The present invention also relates to the use of a 3D microfluidic system as defined above or capable of being prepared by a manufacturing method as defined above to detect and possibly quantify at least one analyte that may be present in a solution of interest or in a gaseous fluid of interest. By "gaseous fluid of interest" is advantageously meant an air sample or a sample from a gaseous industrial effluent.

In other words, the present invention relates to a method for detecting and possibly quantifying at least one analyte that may be present in a solution of interest or in a gaseous fluid of interest, comprising the steps of:

a ₂ ) optionally preparing a 3D microfluidic system capable of detecting said analyte according to a manufacturing method as defined above;

b ₂ ) depositing, on said 3D microfluidic system possibly prepared in step (a ₂ ), said solution of interest or bringing into contact said gaseous fluid of interest with said 3D microfluidic system optionally prepared in step (a ₂₎ ); and

c ₂ ) detect and possibly quantify said analyte possibly present.

The analyte to be detected and possibly quantified is present in the solution of interest or the gaseous fluid such as previously defined can be chosen from the group consisting of a molecule of biological interest; a molecule of pharmacological interest; a toxin; a carbohydrate; a peptide; a protein; a glycoprotein; an enzyme; an enzymatic substrate; a nuclear or membrane receptor; an agonist or antagonist of a nuclear or membrane receptor; a hormone; a polyclonal or monoclonal antibody; an antibody fragment such as an Fab, F (ab ') ₂ , Fv fragment or a hypervariable domain or CDR for "Complementarity Determining Region"; a nucleotide molecule; a pollutant, advantageously organic, water; a pollutant, advantageously organic, of the air; a bacterium and a virus.

The term "nucleotide molecule" used herein is equivalent to the following terms and expressions: "nucleic acid", "polynucleotide", "nucleotide sequence", "polynucleotide sequence". By "nucleotide molecule" is meant, in the context of the present invention, a chromosome; a gene ; a regulatory polynucleotide; DNA, single-stranded or double-stranded, genomic, chromosomal, chloroplast, plasmidic, mitochondrial, recombinant or complementary; total RNA; messenger RNA; a ribosomal RNA (or ribozyme); a transfer RNA; a sequence serving as an aptamer; a portion or a fragment thereof.

The reagent used to functionalize the microfluidic system used in the context of the present invention is any molecule capable of forming with the analyte to detect a binding pair, the reagent and the analyte corresponding to the two partners of this binding pair. . The bonds used in the analyte-reagent link are either non-covalent and low energy bonds such as hydrogen bonds or Van der Waals bonds, or high energy bonds of the covalent bond type.

The reagent used is therefore dependent on the analyte to be detected. Depending on this analyte, the skilled person will know, without inventive effort, choose the most suitable reagent. It may be selected from the group consisting of a probe molecule; a carbohydrate; a peptide; a protein; a glycoprotein; an enzyme; an enzymatic substrate; a membrane or nuclear receptor; an agonist or antagonist of a membrane or nuclear receptor; a toxin; a polyclonal or monoclonal antibody; an antibody fragment such as a Fab fragment, F (ab ') ₂ , Fv or a hypervariable domain (or CDR for "Complementarity Determining Region"); a nucleotide molecule as defined above; a peptide nucleic acid and an aptamer such as DNA aptamer or RNA aptamer. By "probe molecule" is meant, in the context of the present invention, a molecule specific for one (or more) analyte (s) for which contacting with at least one of these analytes results in at least one modification of the spectral properties of this probe molecule.

The probe molecule used in the context of the present invention is a molecule which has fluorogenic or chromogenic properties, that is to say that it becomes fluorescent or stains when it interacts with at least one specific analyte . In general, the interaction of the probe molecule with at least one specific analyte produces a detectable optical signal. The interaction may consist in the creation of irreversible and selective binding, in particular of the covalent bonding type between the probe molecule and at least one specific analyte.

Those skilled in the art know different probe molecules that can be used in the context of the present invention and are known for detecting specific analytes such as an adhéhyde, formaldehyde, acetaldehyde, naphthalene, a primary amine, especially an aromatic amine, indole , skatole, tryptophan, urobilinogen, pyrrole, benzene, toluene, xylene, styrene or napthalene. Such probe molecules are chosen in particular from the enaminomes and the corresponding β-diketone / amine pairs, the imines and the hydrazines, the 4-aminopent-3-en-2-one, the croconic acid and the probe molecules. aldehyde function are p-dimethylaminobenzaldehyde, p-dimethylaminocinnamaldehyde, p-methoxybenzaldehyde and 4-methoxy-1-naphthaldehyde, mixtures and salts derived from these compounds.

The use of a 3D microfluidic system according to the invention or capable of being prepared by a manufacturing method according to the present invention for detecting and possibly quantifying at least one analyte as defined above, thus finds applications in the field of biochemistry, microbiology, in the field of medical diagnosis, in the field of nuclear field, in the field of quality control, in the field of agro-food, in the field of the detection of illicit substances, in the field of defense and / or biodefense; in the field of veterinary, environmental and / or sanitary control and / or in the field of perfumery, cosmetics and / or aromas.

Step (a ₂ ), when it is to be implemented, consists in preparing a 3D microfluidic system capable of detecting at least one given analyte, ie a 3D microfluidic system comprising at least one reagent capable of recognizing said analyte in a specific manner. .

During step (b ₂ ) of the method according to the present invention, depositing the solution of interest consists in depositing one (or more) drop (s) of said solution of interest at one (or more) ) channel (channels) fluidic (s) present (s) on the surface of the system muicrofluidic 3D. The volume of solution of interest deposited on each fluidic channel is between 1 μΙ and 10 ml, especially between 20 μΙ and 5 ml, in particular between 400 μΙ and 2 ml.

Step (c ₂ ) of the method according to the present invention consists in detecting and possibly quantifying the signal emitted by

- the analyte,

the binding pair formed between said analyte and the reagent present in the 3D microfluidic system possibly prepared in step (a ₂ ),

a secondary reagent capable of recognizing the analyte or the binding pair formed between said analyte and the reagent.

The secondary reagent may be selected from the group consisting of a carbohydrate; a peptide; a protein; a glycoprotein; an enzyme; an enzymatic substrate; a membrane or nuclear receptor; an agonist or antagonist of a membrane or nuclear receptor; a toxin; a polyclonal or monoclonal antibody; an antibody fragment such as a Fab fragment, F (ab ') ₂ , Fv or a hypervariable domain (or CDR for "Complementarity Determining Region"); a nucleotide molecule such as previously defined; a peptide nucleic acid and an aptamer such as DNA aptamer or RNA aptamer.

For the purpose of detection, the analyte and / or the secondary reagent has at least one easily detectable group, ie a group which emits a measurable signal (enzymatic, fluorescent, chromogenic, radioactive, etc.) or which allows the formation of a measurable signal.

In the ^era form of implementation of the easily detectable moiety, the latter may be an enzyme or a molecule capable of generating a detectable signal and optionally quantifiable under special conditions as when the power in the presence of a suitable substrate. By way of illustrative and nonlimiting examples, mention may be made of biotin, digoxygenine, 5-bromodeoxyuridine, alkaline phosphatase, peroxidase, glucose amylase and lysozyme.

In a ^2nd form of implementation of the easily detectable moiety, it may be a fluorescent label, chimiofluorescent or bioluminescent such as cyanine and derivatives thereof, fluorescein and its derivatives, rhodamine and its derivatives, GFP (for " Green Fluorescent Protein ") and its derivatives, coumarin and its derivatives and umbelliferone; luminol; luciferase and luciferin.

In a ^3rd form of implementation of the easily detectable moiety, the latter may be a marker or radioactive isotope or an organic group containing at least one marker or radioactive isotope. Such a marker or radioactive isotope is especially iodine-123, iodine-125, iodine-126, iodine-133, iodine-131, iodine-124, indium-111, indium-113m, bromine-77, bromine-76, gallium-67, gallium-68, ruthenium-95, ruthenium-97, technetium-99m, fluorine-19, fluorine-18 , carbon-13, nitrogen-15, oxygen-17, oxygen-15, oxygen-14, scandium-47, tellurium-122m, thulium-165, yttrium-199 , copper-64, copper-62, carbon-11, Pazote-13, gadolidium-68 and rubidium-82. In a 4 ^th form of implementation of the easily detectable moiety, the latter may be a paramagnetic or ferromagnetic marker such as the N-oxide 2,2,6,6-tetramethylpiperidine (TEMPO) or capable of chelating paramagnetic ion complex or ferromagnetic such as Gd ³⁺ , Cu ²⁺ , Fe ³⁺ , Fe ²⁺ and Mn ²⁺ .

In a ^fifth form of implementation of the easily detectable group, the latter may be a gold particle or a dense compound such as a nanoparticle and in particular, by way of example, a (nano) ferric oxide particle or gold.

Thus, the detection during step (c ₂ ) according to the invention can be carried out in particular by colorimetric, (chemo) fluorescence, bioluminescence, radioactive, ferromagnetic, paramagnetic and / or electrical measurements.

Advantageously, this detection is carried out by measuring the variation of absorbance, (chemo) fluorescence, (bio) luminescence, radioactivity, ferromagnetism or paramagnetism of the 3D microfluidic system according to the present invention or at least one of the spots. it presents when this system is brought into contact with a solution of interest containing the analyte (s) as (s) previously defined (s). By way of examples, when the detection is carried out by optical measurement, the wavelength for which the absorbance, (chemo) fluorescence or (bio) luminescence of the reagent such as a probe molecule is preferably chosen is or the product formed by the interaction or reaction between the reagent and the detected analyte is the highest.

As a variant, this detection is carried out by measuring the variation of the electrical signal emitted by the 3D microfluidic system according to the present invention or at least one of the spots that it presents when this system is brought into contact with a solution of interest containing the analyte (s) as previously defined. The electrical and possibly optical, colorimetric detection of (chemo) fluorescence, bioluminescence, radioactive, ferromagnetic or paramagnetic may require that the 3D microfluidic system according to the present invention be deposited on a detection system including electrical tracks, in particular copper or copper. or or that the detection stage of the 3D microfluidic system according to the present invention comprises electrical tracks including copper or gold, protected from oxidation, in particular by the deposition of parylene. These electrical connectors can be used for electrical detections or to integrate more complex systems such as diodes to stimulate fluorescence or phosphorescence of the reagent such as a probe molecule, or the product formed by the interaction or the reaction between the reagent and the analyte detected at the detection site.

Whatever the nature of the measurement carried out, the latter can be compared to the measurement obtained from calibrated solutions of known analytes to directly give information on the quantity and / or the nature of the analyte (s). content (s) in the solution of interest.

In a particular embodiment, the exposure of the 3D microfluidic system according to the present invention to a liquid solution can give rise to a visual, photographic or scanner observation. In this case, a comparison of the intensity of the staining developed with those previously established during a calibration for a predefined range of concentrations, allows to roughly deduce the concentration of the analyte. For fine quantitative measurement, a measurement of the absorption variation at a point or wide spectral range as a function of the contact time is required to determine a rate of formation of the colored complex. The value of this speed will be compared to those obtained for a standard range under the same conditions.

Before or during the detection step (c ₂ ), solutions other than the solution of interest or that the gaseous fluid may have to be deposited on the 3D microfluidic system according to the present invention. Indeed, washing solutions to eliminate any non-specific binding involving the reagent, a solution containing the secondary reagent or solutions containing an ada pte substrate to an enzyme carried by the analyte or the secondary reagent may have to be deposited on the system. 3D microfluidic according to the invention.

The present invention finally relates to the use of a 3D microfluidic system as defined above or capable of being prepared by a manufacturing method as defined above for purifying an analyte present in a solution of interest or in a gaseous fluid. 'interest. In other words, the present invention relates to a method for purifying at least one analyte present in a solution of interest or a gaseous fluid of interest, comprising the steps of:

a ₃ ) optionally preparing a 3D microfluidic system capable of purifying said analyte according to a manufacturing method as defined above;

b ₃ ) depositing, on said 3D microfluidic system optionally prepared in step (a ₃ ), said solution of interest or bringing into contact said gaseous fluid of interest with said 3D microfluidic system optionally prepared in step (a ₃ ); and

c ₃ ) purifying said analyte.

All that has been described, as part of the method of detection and possible quantification, as for step (a ₂ ), step (b ₂ ), the analyte to be detected and possibly quantified and to the solution of interest also applies mutatis mutandis to step (a ₃ ), step (b ₃ ), the analyte to be purified and the solution of interest in the purification process according to the present invention.

Indeed, in a first ^embodiment , the purification process according to the invention may require the use of a microfluidic system comprising at least one reagent capable of recognizing the analyte at purify in a specific way. The purification in step (c ₃ ) is carried out by recovering the analyte from the binding pair formed by the latter and the reagent.

In a 2 ^nd form of implementation, the method of purification according to the invention does not implement a specific reagent for the analyte as defined above. But in this form of implementation, the purification is carried out by progressive elimination of the constituents of the solution of interest, other than the analyte to be purified. Thus, step (a ₃ ), when it is to be implemented, consists of preparing a 3D microfluidic system of which one (or more) layer (s) comprises (include) one (or more) suitable compound (s). (s) trapping one (or more) of the components of the solution of interest, other than the analyte to be purified. In this form of implementation, step (c ₃ ) of the method consists in recovering the analyte at the level of at least one lower layer of the 3D microfluidic system.

In the context of the use of the 3D microfluidic system according to the present invention in the field of purification, porous polymer membranes having anion or cation exchange groups can be used to carry out a purification by chromatography-exchange chromatography. ions. It is also possible to use porous polymeric membranes or other porous materials previously modified by specific organic or biological molecules in order to perform affinity chromatography.

Other features and advantages of the present invention will become apparent to those skilled in the art upon reading the examples below given for illustrative and non-limiting purposes, with reference to the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of different support parts, usable in a 3D microfluidic system according to the invention. Figure 2 is a schematic diagram of different parts of the fluidic channel type, usable in a 3D microfluidic system according to the invention.

Figure 3 is a diagram of a microfluidic system according to the invention with the various layers constituting it, the drop symbolizing the solution of interest to be analyzed or purified and the arrow the direction of migration of the sample.

Figure 4 shows a housing in which the 3D microfluidic system according to the invention can be inserted.

Figure 5 shows results obtainable for different solutions of interest (Figures 5B, 5C, 5D, 5E and 5F) using a microfluidic system according to the invention as described in Figure 5A.

Figure 6 shows the various stages (Figure 6A, 6B and 6C) with the 2 ^nd portion fitted into the bracket ^1, before assembly of a 3D microfluidic system 3 stages (or layers).

Figure 7 shows the different stages (Figures 7A, 7B, 7C, 7D and

7E) with the (or) ^{2nd (s>} portion (s) nested (s) in the carrier ¹ prior to assembly of a 3D microfluidic system 5-stage (or layers).

Figure 8 shows a global system after assembly with Figure 8A showing the top of the system at which is introduced the solution of interest to be analyzed and Figure 8B the reading stage of the results.

Figure 9 is a schematization of a microfluidic system according to the invention used for the detection of botulinum toxin A (Figure 9A) and the detection layer of this system (Figure 9B).

Figure 10 shows the results obtained with a solution of interest containing (Figure 10B) or not (Figure 10A) botulinum toxin A.

Figure 11 is a layer-by-layer representation of porous hydrophobic material elements and elements of porous hydrophilic material, alone or together, constituting a 3D microfluidic system with integrated reservoir and double spiral. Figure 12 is a representation of the integrated, dual-spiral, 3D microfluidic system obtained from the elements shown in Figure 11.

Figure 13 is a schematic of the migration of the sample into the empty cannula and porous hydrophilic channels of the microfluidic system of Figure 12.

Figure 14 is a top view of the microfluidic system with integrated reservoir and double spiral (layer (1)), once a sample of interest introduced into the latter.

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

I. Preparation of a 3D microfluidic system according to the invention.

The 3D microfluidic system according to the present invention described below is a system suitable for hydrophilic solutions of interest.

1.1. Preparation of the support parts of the system layers.

FIG. 1 shows the "negatives" (1, 3, 5, 7 and 9) made in a PET type polymer sheet with one or two recesses (s) per negative, of identical or different shapes, obtained after manual cutting type stamping or by C0 ₂ laser printer.

Three of these "negatives" form the support portions as previously defined of the layers of the system (see Figure 3).

1.2. Preparation of the "fluidic channel" type parts of the system layers.

Figure 2 shows the "positives" (10, 11 and 12) made in a sheet of paper or fiberglass, obtained after manual cutting type drawing or C0 ₂ laser printer. These 3 types of "positives" form the fluidic channel parts that fit into the recesses of the "negatives" prepared in 1.1.

1.3. Assembly of the system and system thus obtained.

The assembly of the device is done by:

1 / interlocking the hydrophilic cut piece ("positive") in the hydrophobic material ("negative"), the assembly constituting a mixed layer of the system according to the invention;

2 / bonding on the surface of this mixed layer of the double-sided adhesive cut to leave recesses in the fluidic channel;

3 / positioning a new mixed layer on the surface of the adhesive.

Steps 2 and 3 can be repeated as many times as necessary. A 3D microfluidic system that can be obtained from

"Negatives" and "positives" in Figures 1 and 2 is shown in Figure 3.

The layers (1) and (9) of this system do not constitute layers with a ^lere and at least a 2 ^nde parts as defined in the present invention. Indeed, the recesses of the support part are left as is, without a "positive" is nested. On the other hand, the layers (3), (5) and (7) in which a "positive" (10) is respectively nested, two "positive" (11) and two "positive" (12) are indeed layers with a l ^st and at least one 2 ^nd parts as defined in the present invention.

The layers (2), (4), (6) and (8) represent the double-sided Scotch ^® respectively placed between the layers (1) and (3), (3) and (5), (5) and (5). 7) and (7) and (9). These layers have recess areas to ensure the continuity of the fluidic channel. Some of these recesses are left as layers (2) and (8), while in the others, nested "positives" material hydrophilic. Thus, two "positives" (11) are nested in the layers of Scotch ^® (4) and (6).

In the microfluidic system shown in FIG. 3, the layers of hydrophobic material have clearly defined functions with:

the layer (1) which is the layer on which the sample of the solution of interest to be analyzed or purified represented by a drop is deposited;

- The layer (3) which is a layer for dividing the sample, in the case presented, in two;

the layers (5) and (7) corresponding to the analysis stages; and - the layer (9) which participates in supporting the hydrophilic parts of the upper stages and in particular of the stage (7).

All of these layers are deposited on absorbent paper to maintain migration and the entire device is inserted into a small plastic box as shown in Figure 4.

II. Example of a 3D microfluidic system according to the multiparameter invention.

FIG. 5A presents a microfluidic system according to the invention with 4 pads, of different shapes facilitating in particular the reading of the results:

- with a control pad;

a block whose part of fluidic channel type present in the analysis layer of anti-anthrax antibodies and thus able to reveal, in the solution of interest to be analyzed, the presence of Anthrax "Plot Anthrax";

a pad whose part of fluidic channel type present in the anti-ricin antibody analysis layer and thus able to reveal the presence of ricin "Plot Ricin" with in the hydrophilic part; and - A stud whose fluidic channel type part in the analysis layer of botulinum toxin A antibodies and thus able to reveal the presence of botulinum toxin A "Botulinum Toxin A".

Figures 5B, 5C, 5D, 5E and 5F show the theoretical result obtained for a negative sample for the 3 agents tested; a positive sample for botulinum toxin A; a positive sample for anthrax; a positive sample for anthrax and ricin; and a sample made under unsuitable conditions. III. Examples of development of a system having a multi-pin reading stage.

I II .1. Reading floor with 4 studs.

The various stages forming the pa rt ^ere the support consist of PET sheet 100 microns thick. The different stages were obtained after cutting with a C0 ₂ laser (Laser GCC Pro Spirit 25 watts). The design was done on the Corel Draw 5 software.

The 2 ^nd portion (flow system) was obtained after cutting pa per Whatman CF1 by a C0 ₂ laser (Laser CCG Pro Spirit 25 watts). The design was done on the Corel Draw 5 software.

Figure 6 shows the different stages (Figure 6A, 6B and 6C) with the

^2nd part fitted into the assembly ¹ before support. In particular,

FIGS. 6A, 6B and 6C respectively correspond to the distribution, distribution and analysis stages of the solution of interest.

I II .2. Reading floor with 16 pins.

The various stages forming the pa rt ^ere the support consist of PET sheet 100 microns thick. The different stages were obtained after cutting with a C0 ₂ laser (Laser GCC Pro Spirit 25 watts). The design was done on the Corel Draw 5 software. The 2 ^nd portion (flow system) was obtained after paper cutting Whatman CF1 by a C0 ₂ laser (Laser CCG Pro Spirit 25 watts). The design was done on the Corel Draw 5 software.

Figure 7 shows the different stages (Figures 7A, 7B, 7C, 7D and 7E) with the (s) 2 ^nd part (s) nested in the 1 ^st support before assembly. In particular, FIGS. 7A, 7B, 7C, 7D and 7E respectively correspond to stages of distribution, distribution, distribution, distribution and analysis of the solution of interest. III.3. Reading stage for 16-point pH.

The various stages constituting the ^ere partie support consist of PET sheets 100 μιη thick. The different stages were obtained after cutting with a C0 ₂ laser (Laser GCC Pro Spirit 25 watts). The design was done on the Corel Draw 5 software.

The 2 ^nd portion (flow system) was obtained after pH Whatman paper cutting by a laser C0 ₂ (Laser CCG Pro Spirit 25 watts). The design was done on the Corel Draw 5 software.

Figure 8 shows a global system after assembly. The figure

8A shows the top of the system at which the solution of interest to be analyzed is introduced and Figure 8B shows the reading stage of the results.

IV. Chemical modification of cellulose for the covalent grafting of biomolecules.

IV.1. Preliminary remarks.

The 3D microfluidic systems according to the invention can incorporate the reading stage of the system.

To do this, the 2 ^nd portion (ie the fluid system that can be of the cellulose, for example) must be modified in order to graft it, so covalently, biolomecules or organic molecules of interest that will obtain the information that is to be extracted from the solution of interest.

The person skilled in the art knows that it is extremely difficult to modify the cellulose. These modifications generally take place on cellulose in the form of fibrils under conditions incompatible with the living (organic solvent and high temperatures). In addition, it is almost impossible to work on shaped cellulose, for example, in the form of a sheet without destroying the macroscopic structure of the material.

In order to circumvent these problems, the inventors have developed methods for modifying, under conditions compatible with living matter, cellulose in the form of leaves without altering the macroscopic structure. Two synthetic routes have been used, the first based on a solution reduction of diazonium salts for which the skilled person can find more information in the international application WO 2008/078052 [7] and the other based on a photochemical reaction at 385 nm using arylazide derivatives. Each of these routes can be carried out preferentially in water or in biological buffer solutions but can also be used in solvent mixing systems or in organic solvent. These two routes have made it possible to introduce, at the level of the cellulose sheet, functions making it possible to graft biological molecules or organic molecules under biologically compatible conditions, of the carboxylic acid, amine or thiol type.

In the same way as described in the international application WO 2008/078052 [7], a polymer having a function of interest in the context of the present invention can be grafted, covalently, in cellulose without destroying its macroscopic format. and in conditions compatible with the living. After modification of a cellulose sheet by these chemical processes, the grafting of biomolecules is carried out under conditions compatible with biology, well known to those skilled in the art.

It is understood that these methods can also be used for the modification of intermediate stages of the system, if, for example, there is a need to purify in an "active" manner (ie a purification via, inter alia, antibody / antigen recognition and not by any process of "passive" type such as the size of the porosity which allows a mechanical purification) or to introduce any group necessary for the proper functioning of the 3D microfluidic system such as, for example, the introduction of anionic or cationic groups for obtain, from cellulose, an equivalent of a Sephadex resin.

IV.2. Grafting of groups.

A. Grafting Polyacrylic Acid onto a Sheet of Paper (Example

AT).

A GraftFast ™ solution for grafting acrylic acid is created. For more information, the experimenter may refer to international application WO 2008/078052 [7].

A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF1 from Whatman. After 1 to 6 hours, the paper is then sintered with milliQ water (10 mL), then with 1 M sodium hydroxide solution (10 mL) and finally with milliQ water. (10mL) before being reacidified with 1M HCl solution (10mL). The paper is then dried in the air. The ATR FTI R analysis clearly shows the presence of the vibration band at 1710 cm ^-1 corresponding to the vibration of the carboxylic acid group.

B. Grafting a poly (carboxyl) phenylene layer onto paper (Example B). 4 aminobenzoic acid (Sigma Aldrich) (2 mmol, 274.3 mg) is converted into a diazonium salt by adding this compound in a solution of HBF ₄ at 48% in the presence of 1.1 equivalents of NaN0 ₂ (2.2 mmol, 151.8 mg) relative to the aniline derivative. The diazonium salt is obtained after precipitation in ether and filtration. The corresponding diazonium salt is dissolved in a solution of HCl at 0.25 N and then 2 mL of H ₃ PO ₂ at 50% by weight are introduced in order to provoke reduction of diazonium salts in solution (for more information, see [7]).

A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF3 from Whatman. After 1 to 6 hours, the paper is then rinsed on sintered with milliQ water (10 mL), then with 1 M sodium hydroxide solution (10 mL) and finally with water. milliQ water. (10mL) before being reacidified with 1M HCl solution (10mL). The paper is then dried in the air.

C. Grafting a poly (amino) phenylene layer on paper (Example C).

1,4-Benzenediamine (Sigma Aldrich) (2 mmol, 216.3 mg) is converted into a diazonium salt by adding this compound in a solution of HBF ₄ at 48% in the presence of 1.1 equivalents of NaN0 _2. (2.2 mmol, 151.8 mg) relative to the aniline derivative. The diazonium salt is obtained after precipitation in ether and filtration.

The corresponding diazonium salt is dissolved in a solution of HCl at 0.25 N and then 2 mL of H ₃ PO ₂ at 50% by weight are introduced in order to cause the reduction of the diazonium salts in solution (for more information, see [7]).

A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF3 from Whatman. After a time of 1 to 6 hours, the paper is then rinsed on a sintered by milliQ water (10 m L). then with a 1M sodium hydroxide solution (10 mL) and then with milliQ water (10 mL) and then reacidified with a solution of 1M HCl (10 mL). The paper is then dried in the air.

D. Grafting a layer of poly (ethyl carboxyl) phenylene on paper (Example D).

3- (4-aminophenyl) propionic acid (Sigma aldrich) (2 m mol, 330.4 mg) is converted into a diazonium salt by adding this compound in a solution of HBF ₄ at 48% in the presence of 1 1 equivalent of NaNO ₂ (2.2 mmol, 151.8 mg) relative to the derivative of niline. The diazonium salt is obtained after precipitation in ether and filtration.

A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF3 from Whatman. After 1 to 6 hours, the paper is then rinsed on a sintered with milliQ water. (10 ml) then with 1 M sodium hydroxide solution (10 ml) and then with milliq water (10 ml) and then reacidified with 1 M HCl solution (10 ml). The paper is then dried in the air.

E. Grafting a layer of poly (ethylamino) phenylene on paper (Example E).

4- (2-aminoethyl) aniline (Sigma Aldrich) (2 mmol, 272.4 mg) is converted to the diazonium salt by adding this compound in a solution of HBF ₄ at 48% in the presence of 1.1 equivalents Na NO ₂ (2.2 mmol, 151.8 mg) relative to the aniline derivative.

The diazonium salt is obtained after precipitation in ether and filtration. The corresponding diazonium salt is dissolved in a solution of HCl 0.25 N then 2 m L of H ₃ PO ₂ at 50% by weight are introduced to cause the reduction of diazonium salts in solution (for more information, see [7]).

A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF3 from Whatman. After a time of 1 to 6 hours, the paper is then rinsed on sintered with milNQ water (10 mL) then with 1 M sodium hydroxide solution (10 mL) and then with milNQ water (10 mL). mL) and then reacidified with 1 M HCl solution (10 mL). The paper is then dried in the air. F. Grafting a layer of poly (thiol) phenylene on paper

(Example F).

4-Aminobenzenethiol (Sigma Aldrich) (2 mmol, 250.4 mg) is converted into a diazonium salt by adding this compound in a solution of HBF ₄ at 48% in the presence of 1.1 equivalents of NaNO ₂ ( 2.2 mmol, 151.8 mg) relative to the aniline derivative. The diazonium salt is obtained after precipitation in ether and filtration.

A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF3 from Whatman. After a time of 1 to 6 hours, the paper is then rinsed on a sintered with milliQ water (10 m L) then with 1 M sodium hydroxide solution (10 mL) and then with milliQ water (10 mL). mL) and then reacidified with 1 M HCl solution (10 mL). The paper is then dried in the air.

G. Grafting of a layer of aminoaryl on paper (Example G). 1,4-Benzenediamine (Sigma Aldrich) is converted into a diazonium salt by adding this compound in a solution of 48% HBF ₄ in the presence 1.1 equivalents of NaNO ₂ (2.2 mmol, 151.8 mg) relative to the aniline derivative. The diazonium salt is obtained after precipitation in ether and filtration.

The diazonium salt is then resuspended in ether cold (4 ° C) and then 4 equivalents of NaN ₃ (4 mmol, 260 mg) are added and the solution is returned to the temperature for 3 h. The resulting solution is filtered in order to get rid of the salts. The ether is evaporated to dryness to give the corresponding arylazide.

The latter is dissolved in water or in a biological buffer such as PBS or TRIS protected from light. A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF3 or CF1 from Whatman. The paper is then subjected to irradiation at 385 nm for a time varying from 1 to 5 min. The paper is then rinsed on a sintered by milliQ water. (10 mL) then with 1 M sodium hydroxide solution (10 mL) and finally with milliQ water (10 mL) before being reacidified with 1 M HCl solution (10 mL). The paper is then dried in the air.

H. Grafting of a carboxylaryl layer on paper (Example H). The 4-aminobenzoic acid (Sigma Aldrich) is converted into a diazonium salt by adding this compound in a solution of HBF ₄ at 48% in the presence of 1.1 equivalents of NaNO ₂ (2.2 mmol, 151.8 mg) relative to the aniline derivative. The diazonium salt is obtained after precipitation in ether and filtration. The diazonium salt is then resuspended in ether at cold (4 ° C.) and then 4 equivalents of NaN ₃ (4 mmol, 260 mg) are added. and the solution returns to temperature for 3 h. The resulting solution is filtered in order to get rid of the salts. The ether is evaporated to dryness to give the corresponding arylazide.

J. Grafting a layer of ethylaminoaryl on paper (Example J). The 4- (2-aminoethyl) aniline (Sigma Aldrich) is converted into a diazonium salt by adding this compound in a 48% solution of HBF ₄ in the presence of 1.1 equivalents of NaNO ₂ (2.2 mmol , 151.8 mg) relative to the aniline derivative. The diazonium salt is obtained after precipitation in ether and filtration.

The latter is dissolved in water or in a biological buffer such as PBS or TRIS protected from light. A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF3 or CF1 from Whatman. The paper is then subjected to irradiation at 385 nm for a time varying from 1 to 5 min. The paper is then rinsed on sintered with milliQ water (10 mL) then with 1 M sodium hydroxide solution (10 mL) and finally with milliQ water (10 mL) before being reacidified with a solution of 1M HCl (10 mL). The paper is then dried in the air. K. Grafting a layer of ethylcarboxylaryl on paper (Example

K).

3- (4-aminophenyl) propionic acid (Sigma Aldrich) is converted into a diazonium salt by adding this compound in a solution of HBF ₄ at 48% in the presence of 1.1 equivalents of NaN0 ₂ (2.2 mmol, 151.8 mg) relative to the aniline derivative. The diazonium salt is obtained after precipitation in ether and filtration.

The corresponding arylazide is dissolved in water or in a biological buffer such as PBS or TRIS protected from light. A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF3 or CF1 from Whatman. The paper is then subjected to irradiation at 385 nm for a time varying from 1 to 5 min. The paper is then rinsed on sintered with milliQ water (10 mL) then with 1 M sodium hydroxide solution (10 mL) and finally with milliQ water. (10 mL) before being reacidified with 1 M HCl solution (10 mL). The paper is then dried in the air.

L. grafting of a layer of thioaryl on paper (Example L).

4-Aminobenzenethiol (Sigma Aldrich) is converted into a diazonium salt by adding this compound in a 48% solution of HBF ₄ in the presence of 1.1 equivalents of NaNO ₂ (2.2 mmol, 151.8 mg ) relative to the derivative of aniline. The diazonium salt is obtained after precipitation in ether and filtration.

The diazonium salt is then resuspended in cold ether (4 ° C.) and then 4 equivalents of NaN ₃ (4 mmol, 260 mg) are added and the solution is dissolved. returns to the temperature for 3 h. The resulting solution is filtered in order to get rid of the salts. The ether is evaporated to dryness to give the corresponding arylazide.

The corresponding arylazide is dissolved in water or in a biological buffer such as PBS or TRIS protected from light. A few drops of the solution thus obtained are deposited on a piece of 2 cm x 2 cm of a sheet of paper CF3 or CF1 from Whatman. The paper is then subjected to irradiation at 385 nm for a time varying from 1 to 5 min. The paper is then rinsed on sintered with milliQ water (10 mL) then with 1 M sodium hydroxide solution (10 mL) and finally with milliQ water (10 mL) before being reacidified with 1M HCl solution (10 mL). The paper is then dried in the air.

IV.3. Grafting of antibodies.

A. on the papers of Examples A, B, QD, E, F, G, H and J.

Anti-ricin antibodies, anti-enterotoxin B from Staphyloccocus

aureus,

antracis, anti-ovalbumin and anti-beta-lactoglobulin in solution in PBS buffer (1 mg / ml) are brought into contact (200 μl) with the papers of Examples A, B, C, D, E, F, G, H and J in the presence of a solution of EDC / NHS (for "1-ethyl-3- (3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide) at 50 mM and are incubated with the papers for 12 h at 4 ° C. . The papers are then rinsed with PBS buffer and then blocked with BSA solution in PBS.

B. on the papers of Examples F and L.

Anti-ricin, anti-enterotoxin B antibodies of Staphylococcus aureus, antacillus antracis, anti-ovalbumin and anti-beta-lactoglobulin in solution in PBS buffer (1 mg / ml) are brought into contact (200 μl) with the papers of Examples F and L and are incubated for 12 h at 4 ° C. The papers are then rinsed with PBS buffer and then they are blocked with a solution of BSA in PBS.

IV.4. Grafting on paper of antibodies modified by an arylazide arm.

Anti-ricin, anti-enterotoxin B, Staphyloccocus aureus, anti-Bacillus antracis, anti-ovalbumin and anti-beta-lactoglobulin antibodies are modified with linkers having an arylazide group such as the ANB-NOS compound or the Sulfo-SANPAH compound ( Pierce, Thermo Fisher) of formula:

The antibodies thus obtained are purified by chromatography. Whatman CF1 paper is impregnated with 100 μl of a solution of these modified antibodies (1 mg / ml) with the arylazide group and then the samples are irradiated at 385 nm for a time varying from 1 to 5 min.

It should be noted that the use of these photoactivatable groups makes it possible not to modify or alter the biomolecules and their biological activity. After the photochemical reaction, the papers are rinsed with PBS buffer (1 mL).

V. Use of a 3D microfluidic system according to the invention for detecting botulinum toxin A.

V.I. System design.

Figure 9A shows the different layers of the 3D microfluidic system according to the invention. Referring to the layers as described for the system of point I, the system implemented for detecting botulinum toxin comprises layers (1) to (5) with the support part of the layers (1), (3) and ( 5) in PET polymer and the portion (s) of the fluidic channel type of the layers (3) and (5) in cellulose or nitrocellulose.

The layers (2) and (4) are made of double-sided Scotch ^® with the recess of the layer (2) left in the state, while in the recesses of the layer (4) have been nested parts of cellulose or nitrocellulose.

As previously described, the polymer sheets and the cellulose sheets are cut in the same pattern, one being the negative of the other. All the leaves are kept using double-sided Scotch ^® .

Finally, as shown in FIG. 9B, the layer (5) corresponding to the analysis stage has fluidic channel portions functionalized, for one, by an anti-botulinum toxin A antibody produced by the CEA and, for the other, hereinafter designated "control plot", by a goat anti-mouse antibody (Jackson Immunoresearch Laboratories Inc., Code 115-005-004). The fluidic part of the analysis stage (ie layer (5)) is in nitrocellulose and the various antibodies have been immobilized on the latter directly, by electrostatic interaction, without any other step than the deposition of the antibody solution.

V.2. Results obtained.

Two different samples were tested on microfluidic systems as prepared in III.1.

¹ sample contains a botulinum toxin anti-mouse antibody

Labeled with colloidal gold (1 μg, antibody and CEPA labeling, Volland et al., 2007 [9])) without botulinum toxin A in 0.1 M potassium phosphate buffer (pH 7.4) + 0.15 M NaCl + 1 mg / ml serum bovine albumin + 0.01% of sodium azide. Figure 10A shows the result obtained after the deposition of a drop of 500 μΙ sample ^1, only the control pad being colored.

The 2 ^nd sample contains a botulinum toxin anti-mouse antibody labeled with colloidal gold (1 mcg; antibody and marking carried out by CEA, Volland et al, 2007 [9].)) And a non-toxic region of the toxin A botulic antibody recognized by the antibody (100 ng / ml, recombinant protein corresponding to the binding domain of botulinum toxin A, the preparation of this recombinant protein being described by Tavallaie et al., 2004 [10]) in phosphate buffer 0.1 M potassium (pH 7.4) + 0.15 M NaCl + 1 mg / ml serum bovine albumin + 0.01% sodium azide. Figure 10B shows the result obtained following the deposition of a droplet of 500 μΙ of the 2 ^nd sample, the two pads being colored.

These results show that the detection of a toxin at the reaction zone is feasible.

VI. Preparation of a 3D microfluidic system with integrated tank and double spiral according to the invention.

The integrated reservoir microfluidic system 3D according to the present invention described below is a system suitable for hydrophilic solutions of interest.

The materials used and the realization of the support parts and the "fluidic channel" type parts are as defined in paragraphs 1.1 and 1.2 above. VI .1. Realization of the integrated tank.

A hollow pattern is made on different stages of the device. This hollow pattern is not subsequently filled with hydrophilic porous material, the stack of stages can create a cavity that is in contact with a hydrophilic porous material. The sample is deposited in the reservoir and then, by capillarity, it migrates into the hydrophilic porous material.

VI.2. Realization of the spiral pattern.

The spiral or double spiral fluidic circuit is cut manually or with a C0 ₂ laser printer:

in the hydrophobic or nonporous matrix, the matrix is recovered with its spiral or double spiral cutout which constitutes the "negative of the device";

in the porous hydrophilic material, the spiral or double spiral cut piece which constitutes the "positive" of the device is recovered and

in the adhesive, the adhesive is recovered with its cutout.

VI.3. Assembly of the system and system thus obtained.

The assembly of the device is done by

1 / interlocking of the hydrophilic piece cut ("positive") in the hydrophobic material ("negative"), the assembly constituting a mixed layer;

2 / gluing on the surface of this mixed layer of the cut double-sided adhesive;

3 / positioning a new mixed layer, a mixed layer comprising at least one unfilled recessed zone or a hydrophobic material support layer, none of which is filled on the surface of the adhesive,

steps 2 and 3 can be repeated as many times as necessary.

The various elements constituting the layers of a 3D microfluidic system with integral reservoir and spiral fluidic circuit and the assembled layers obtained are presented in FIG. 11 and the final microfluidic system is shown in FIG. corresponding to the double-sided scotch ^® possibly present between two layers illustrated in Figures 11 and 12 are not shown on the latter.

The layers (1) and (9) of this system do not constitute layers with a ^lere and at least a 2 ^nde parts as defined in the present invention. Indeed, none of the recesses of the layer (1) is filled by a porous hydrophilic component. This layer (1) is, on the one hand, the layer at which the sample of the solution of interest to be analyzed or purified represented by a drop is introduced via an unfinished circular recess and on the other hand , the layer at which other unconfirmed recesses vis-à-vis the detection zones made on the double spiral of the layer (2) allow the visualization of the signal that reveals or not the presence of the desired agent . Similarly, the support layer (9) is left in the participating state, in fact, in support of the hydrophilic portions of the upper stages.

In addition, the system of Figures 11 and 12 is characterized by

(i) at the level of the layer (2), a double spiral of analysis in porous hydrophilic material at the level of which detection zones have been produced in particular by implementing one or more of the protocols described in paragraph IV;

(i) at the level of the layer (6), a reservoir whose base is formed by a portion of the support of the layer (7) and the walls by the edges of one of the recesses of the layer (6), the the recess of the reservoir, although not filled, having an element made of a porous hydrophilic material, and

(iii) at the level of the layer (8), a hydrophilic matrix corresponds to absorbent paper which maintains the movement of the sample in the device by capillarity and which retains it once the latter has circulated over the entire device, the The hydrophilic matrix of this layer (8) can be considered, therefore, as a "biological bin" or as a low-cost system acting as a "pump". The layers (1), (2), (3), (4) and (5) all have an unfilled recess, the uncompleted recesses of these layers being centered on a common axis and having an identical diameter whereby they form a cannula-type channel for driving the solution sample of interest to be analyzed or purified from the layer (1) to the reservoir of the layer (6).

In addition, the layers (3), (4) and (5) all have a 1 ^st recess filled with a hydrophilic porous material for conveying by capillary action of the sample solution of interest to be analyzed or purifying tank from the layer (6) to the double spiral of the layer (2). The hydrophilic porous material of this recess ¹ filled at the layer (5) is in the extension of the porous hydrophilic material present at the level of the reservoir layer (6). These 1 ^ers recesses do not have identical shapes from one layer to another and are not necessarily centered on a common axis, the only condition being that there is a fluidic continuity between the porous hydrophilic materials that fill them.

Finally, the layers (3), (4), (5), (6) and (7) each have two recesses of identical shape and filled with a porous hydrophilic material, advantageously with the same porous hydrophilic material; these two recesses each in the fluidic continuity of one of the ends of the double spiral of the layer (2) make it possible to route, by capillarity, the sample solution of interest, after it comes into contact with the spiral of material hydrophilic porous layer (2) to the hydrophilic material of the layer (8). These recesses do not have identical shapes from one layer to another and are not necessarily focused on two common axes, the only requirement being that there is a preview ^ere fluidic continuity between the porous hydrophilic materials that meet ¹ of these recesses and a 2 ^nd fluidic continuity between porous hydrophilic materials that fill the 2 ^nd of these recesses.

Thus, in the system shown in Figure 12, the sample of interest is contacted or through several layers multiple times, the time to go ^ere to the reservoir layer (6) (downlink), a 2 ^does time to go from the reservoir layer (6) to the layer (2) analysis (upward direction), and a ^3rd and final times to go from the analysis layer (2) to the layer (8) "trash". Figure 13 schematizes chronologically the movement of the sample in the system of Figure 12.

As explained above, the double spiral of the layer may be functionalized by different reagents or compounds capable of detecting or trapping a constituent or an analyte that may be present in the solution of interest to be analyzed. More particularly, this functionalization with different reagents or compounds takes place at particular zones of the double spiral. FIG. 14 presents the information that can be obtained by looking at the layer (1) of the system of FIG. 12. Thus, from a sample of interest deposited on the system according to the invention which is not divided into two at the level of the double spiral, it is possible to obtain information for 16 constituents or analytes present or not in this sample (16 pads of Figure 14).

BIBLIOGRAPHY

1. International application WO 2009/121037 in the name of the President and Fellows of Harvard College, published on ¹ October 2009.

2. Martinez, A. et al., 2008, "Three-dimensional microfluidic devices fabricated in paper and tape", PNAS, vol. 105, pp. 19606-19611.

3. Li, X. et al. , "Manufacturing of paper-based microfluidic sensors by printing", Colloids and Surfaces B: Biointerfaces, pp. 564-570.

4. Lu, Y. et al., 2010, "Manufacturing and characterization of paper-based microfluidics prepared in nitrocellulose membrane by wax printing", Anal. Chem., Vol. 82, pp. 329-335.

5. International application WO 2011/000047, on behalf of Monash University, published on January 6, 2011.

6. International Application WO 2010/003188, in the name of Monash University, published on January 14, 2010.

7. International application WO 2008/078052, on behalf of the CEA, published on July 3, 2008. 8. International application WO 2009/1211944, on behalf of the CEA, published on October 8, 2009. 9. Volland H., et al., 2008, "A sensitive sandwich enzyme immunoassay for free or complexed Clostridium botulinum neurotoxin type A.", J. Immunol. Methods, vol. 330, pp. 120-129. 10. Tavallaie, M., et al., 2004. "Interaction between the two subdomains of the C-terminal part of the neurotoxin botulinum A is essential for the generation of protective antibodies. FEBS Lett., Vol. 572, page 299.

Claims

1) three-dimensional microfluidic system (or 3D) comprising a plurality of layers stacked on each other, characterized in that at least one of said layers consists of a ^st and the at least one 2 ^nd portions, separate the 'from each other, with the 2 ^nd and wettable porous portion with a solution of interest, previously cut, fitting into a recess of the ^era nonporous portion and / or non-wettable by said solution of interest, said 2 ^nd portion serving as a fluid channel for transferring said solution of interest by capillary action in the plane and / or the thickness of said system.

2) 3D microfluidic system according to claim 1, characterized in that said portion is the ^era:

- porous and hydrophobic;

- non-porous and hydrophilic; or

- non-porous and hydrophobic;

the (or) 2 ^{nd (s)} part (s) is hydrophilic (s) and porous (s).

3) 3D microfluidic system according to claim 1 or 2, characterized in that the said ^ere moiety is selected from the group consisting of a film, porous or nonporous, polyethylene terephthalate (PET); a porous or non-porous membrane of polyethylene (PE); a porous or non-porous polypropylene (PP) membrane; a porous or non-porous film, or a porous or non-porous, fluorine-containing membrane such as a porous or non-porous film, or a porous or non-porous membrane of hydrophobic polyvinylidene fluoride (PVDF); a porous or non-porous membrane of polytetrafluoroethylene (PTFE); a porous or non-porous copolymer film comprising vinylidene fluoride and tetrafluoroethylene; a porous or non-porous copolymer film comprising vinylidene fluoride and hexafluoropropylene; a porous or non-porous film of polymethyl methacrylate (PMMA); a porous or non-porous film of poly (n-butyl acetate); a porous or non-porous film of poly (benzyl methacrylate); a porous or non-porous film of poly (chlorotrifluoroethylene); a porous membrane and, in particular a porous ion exchange membrane, functionalized by hydrophobic groups; a styrenic polymer such as an advantageously porous polystyrene resin; a porous or non-porous film of polyacrylonitrile; a porous or non-porous film of polymethacrylonitrile; a film, porous or nonporous, polyimide such as Kapton ^® and mixtures thereof.

4) 3D microfluidic system according to any one of claims 1 to 3, characterized in that said 2 ^nd portion is selected from the group consisting of paper including cellulose type; cotton paper; agarose; gelatin; cellulose; methylcellulose; carboxymethylcellulose; nitrocellulose; cellulose acetate ester; an alginate; a polyolefin; a porous membrane and, in particular a porous ion exchange membrane, functionalized with hydrophilic groups such as a NAFION membrane; a membrane-type Sephadex resin or a PVDF membrane; a fiberglass fabric; a polyacrylamide gel; a sepharose gel or a mixture thereof.

5) 3D microfluidic system according to claim 1, characterized in that said portion is the ^era:

- porous and hydrophilic;

- non-porous and hydrophilic; or

- non-porous and hydrophobic;

the (or) 2 ^{nd (s)} part (s) being hydrophobic (s) and porous (s). 6) 3D microfluidic system according to claim 1 or 5, characterized in that said ^ere the portion is selected from the group consisting of paper; cotton paper; agarose; gelatin; cellulose; methylcellulose; carboxymethylcellulose; nitrocellulose; cellulose acetate ester; an alginate; a polyolefin; a membrane and, in particular an ion exchange membrane, functionalized, advantageously by radiografting, with hydrophilic groups such as a NAFION membrane; a membrane-type Sephadex resin or a PVDF membrane; a fiberglass fabric; a non-porous film of polyethylene terephthalate (PET); a non-porous polyethylene (PE) membrane; a non-porous polypropylene (PP) membrane; a non-porous film of polyimide such as Kapton ^®; a polyacrylamide gel; a sepharose gel or a mixture thereof. 7) 3D microfluidic system according to any one of claims 1, 5 or 6, characterized in that said 2 ^nd portion is selected from the group consisting of paper rendered hydrophobic by treatment, a porous film of polyethylene terephthalate (PET); a porous polyethylene (PE) membrane; a porous polypropylene (PP) membrane; a porous film or a porous membrane containing fluorine such as a porous film or a porous membrane of hydrophobic polyvinylidene fluoride (PVDF); a porous polytetrafluoroethylene (PTFE) membrane; a porous copolymer film comprising vinylidene fluoride and tetrafluoroethylene; a porous copolymer film comprising vinylidene fluoride and hexafluoropropylene; a polyacrylamide gel; a porous polymethyl methacrylate film (PMMA); a porous film of poly (n-butyl acetate); a porous film of poly (benzyl methacrylate); a porous film of poly (chlorotrifluoroethylene); a porous membrane and, in particular a porous ion exchange membrane, functionalized by groups hydrophobic; a styrenic porous polymer such as a porous polystyrene resin; a porous polyacrylonitrile film; a porous film of polymethacrylonitrile and a mixture thereof.

8) 3D microfluidic system according to any one of the preceding claims, characterized in that at least one layer consisting of a ^1st part and at least a 2 ^nde part, distinct one of the the other, with the 2 ^nd and wettable porous portion with a solution of interest fitting into a recess of the ^era nonporous portion and / or non-wettable by said solution of interest, the said ^ere partie further has at least one uncompleted recess.

9) 3D microfluidic system according to any one of the preceding claims, characterized in that it comprises at least one layer consisting solely of a non-porous support and / or non-wettable by a solution of interest and hollowed out at the level of one or more defined areas.

10) 3D microfluidic system according to any one of the preceding claims, characterized in that it comprises at least one layer of which at least a 2 ^nd part plays the role of biological bin.

11) 3D microfluidic system according to any one of the preceding claims, characterized in that it comprises at least one layer of which at least a 2 ^nde portion has a spiral shape or double spiral.

12) 3D microfluidic system according to any one of the preceding claims, characterized in that it has, between at least two consecutive layers, an element capable of joining these two layers together and / or to ensure the seal between these two layers.

13) 3D microfluidic system according to claim 12, characterized in that said element is in the form of a double-sided Scotch ^® , a stretchable film type Saran microwave stretch film or a derivative of a primary of adhesion supported.

14) Device comprising a 3D microfluidic system as defined in any one of the preceding claims.

15) A method of manufacturing a 3D microfluidic system as defined in any one of claims 1 to 13, said method comprising the steps of:

ai) preparing, in a layer of non-porous material and / or non-wettable by a solution of interest, a recess;

bi) cutting, in a layer of a porous material and wettable by said solution of interest, at least one form consistent with the recess prepared in step (ai);

di) optionally repeat steps (ai), (bi) and (ci); and ei) assembling the different layers of the 3D microfluidic system.

16) A manufacturing method according to claim 15, characterized in that, in said step (ai), said recess has a section relative to the plane of the spiral-shaped layer or double spiral. 17) Use of a 3D microfluidic system as defined in any one of claims 1 to 13 or capable of being prepared by a manufacturing method according to claim 15 or 16 for detecting and possibly quantifying at least one analyte that may be present in a solution of interest or in a gaseous fluid of interest.

18) Use of a 3D microfluidic system as defined in any one of claims 1 to 13 or capable of being prepared by a manufacturing method according to claim 15 or 16 for purifying at least one analyte present in a solution of interest or in a gaseous fluid of interest.

19) Use according to claim 17 or 18, characterized in that said analyte is selected from the group consisting of a molecule of biological interest; a molecule of pharmacological interest; a toxin; a carbohydrate; a peptide; a protein; a glycoprotein; an enzyme; an enzymatic substrate; a nuclear or membrane receptor; an agonist or antagonist of a nuclear or membrane receptor; a hormone; a polyclonal or monoclonal antibody; an antibody fragment such as an Fab, F (ab ') ₂ , Fv fragment or a hypervariable domain or CDR for "Complementarity Determining Region"; a nucleotide molecule; a pollutant, advantageously organic, water; a pollutant, advantageously organic, of the air; a bacterium and a virus.